Key processes in development, including differential cell proliferation, programmed cell death, migration and cell-cell interactions are recapitulated in a pathologic manner during oncogenesis and metastasis, suggesting an aberrant regulation or 're-activation' of such processes. For example, FGF, Wnt, and Sonic Hedgehog (Shh) signaling pathways are all involved in the genesis of certain human neoplasias. Understanding how developmental processes are normally regulated will be invaluable in deciphering tumor biology and ultimately help to identify new ways to intercept cellular targets that drive tumor cell behavior. My lab identified several new transcription factors (homeobox genes and T-box genes) that appear to play roles in both the formation and pattern of the primary embryonic axis and the limb axis in vertebrate embryos. These transcription factors function as upstream and/or downstream regulators of the Shh and Fgf signaling pathways, which regulate cell survival and proliferation in many contexts in both embryo and adult. Analyzing the role that these transcriptional regulators play in different developmental contexts will provide new insights into the sorts of basic processes that they govern in the cell. We are analyzing the normal developmental functions of several regulators with the long-term aim of linking steps from initial differences in patterns of gene expression to ultimate differences in tissue morphology and structure in the embryo. In this context, potential roles in oncogenesis and other pathologic conditions are also being considered. 1. Proximo-distal (PD) limb bud outgrowth in response to FGF and Wnt signals: 1A. Limb Initiation: functional analysis of T-box transcription factors T (Brachyury) and Tbx5 using sh-RNA and ChIP-Chip approaches. The initiation of limb bud formation and induction of the apical ectodermal ridge (AER) that directs subsequent limb outgrowth appear to be regulated by a cascade of FGF and Wnt signals. Intermediate target genes in this signal relay are largely unknown, but mutant analyses have demonstrated that the related limb-type specific T-box factors Tbx5 in forelimb, and Tbx4 in hindlimb are essential for initiating outgrowth and interact with both FGF and Wnt pathways. Using retroviral misexpression in chick embryos, we previously showed that the prototype T-box factor, T, also regulates AER maturation and maintenance, appearing to function downstream of both FGF and Wnt signals. Mouse embryo null mutants in the T gene die at E10.5 with severe body axis truncations, limiting the ability to analyze development of their limbs, but they do form forelimb buds which are small and have abnormal, poorly developed AERs. We are generating a conditional knock-down approach for analyzing T function in limb development (see part 3B below) to circumvent the problem of early embryonic lethality in conventional mutants, and we are evaluating whether Tbx5/4 and T cooperate to regulate AER formation and outgrowth, or function sequentially (T downstream). In parallel studies, we are developing chromatin immunoprecipitation (ChIP) assays in embryos to allow direct identification of target promoters in vivo for T-box genes that regulate limb outgrowth. We have successfully piloted ChIP assays with embryo limb bud lysates using anti-Tbx5 (see part 3C below). Identification of additional Tbx5 targets in the early limb bud will also provide new insights on key steps in initiation of limb outgrowth. A similar approach can then be used for other T-box genes such as T, for which we have also developed high-specificity polyclonal antibodies. 1B. Gnot1 homeobox function in chick limb: modulator of the pacing of PD outgrowth and scalar expansion in response to FGF signals.
